The hemagglutinin (HA) of influenza virus is the major surface antigen and is one of the best characterized membrane glycoproteins. It has receptor-binding and fusion activity, which are both necessary for the initiation of viral infection. The protein contains a large ectodomain that carries receptor and fusion activities, a stretch of hydrophobic amino acids which constitutes the transmembrane domain, and a short cytoplasmic tail. This cytoplasmic tail contains 10-11 amino acids, depending on the subtype of the HA (Ward, 1981, Curr. Top. Microbiol. Immunol. 94/95:1-74; FIG. 1). Comparison of the HA sequences in this region reveals that 5 amino acids are highly conserved and that they are identical in 11 of the 14 known subtypes (Doyle et al., 1985, J. Cell Biol. 100:704-714; Kawaoka et al., 1990, Virology 179:759-767; Nobusawa et al., 1991, Virology 182:475-485; Simpson & Lamb, 1992, J. Virol. 66:790-803). Two of these conserved residues are cysteines (positions 560 and 563) and a third highly conserved cysteine is located in the membrane-anchoring domain (position 553) (FIG. 1). It as previously been shown that the conserved cysteines of subtype H2, H3 and H7 HAs are post-translationally modified by covalent addition of palmitic acids (Naeve & Williams, 1990, EMBO J. 9:3857-3866; Naim et al., 1992, J. Virol. 66:7585-7588; Schmidt & Lambrecht, 1985, J. Gen. Virol. 66:2635-2647; Steinhauer et al., 1991, Virology 184:445-448). The levels of palmitylation have been quantified for the A/Japan/305/57 HA (H2), whose 17 carboxy-terminal amino acids are identical to those of the A/WSN/33 HA (H1). The cysteines at positions 560 and 563 (FIG. 1) appear to be highly modified, with position 563 incorporating at least half of the fatty acid label, whereas position 553 incorporates only about 10% of the total palmitate (Naim et al., 1992, J. Virol. 66:7585-7588).
The extent and significance of palmitylation of viral proteins is not yet fully understood (reviewed in McIlhinney, 1990, TIBS 15:387-391). The fact that HeLa cells are refractory to influenza virus growth has been ascribed to a defect in palmitylation of this cell line, but other mechanisms responsible for the abortive infection in HeLa cells cannot be ruled out (Portincasa et al., 1992, Res. Virol. 143:401-406). Experiments using hydroxylamine to remove lipid from viral proteins have suggested that the fatty acid moiety is important for membrane fusion (Schmidt & Lambrecht, 1985, J. Gen. Virol. 66:2635-2647). Another report used the antibiotic cerulenin to inhibit acylation and has implicated the lipid in viral release (Schlesinger & Malfer, 1982, J. Biol. Chem. 257:9887-9890). However, these interpretations are not definitive since hydroxylamine might affect the protein structure and cerulenin is known to exert a general toxic effect. For vesicular stomatitis virus (VSV) it has been reported that the elimination of the palmitylation site in the G protein has no effect on membrane fusion or glycoprotein incorporation into virions (Whitt et al., 1989, J. Virol. 63:3569-3578). Studies with alphaviruses have shown that the elimination of either one of the two palmitylation sites in the carboxy terminus of the glycoprotein E2 decreases the efficiency of virus budding (Ivanova & Schlesinger, 1993, J. Virol. 67:2546-2551), and that a mutant with changes in both palmitate addition sites was not viable (Gaedigk-Nitschko & Schlesinger, 1991, Virology 183:206-214).
There is one report which suggests that the A/Japan/305/57 HA (H2) requires palmitate for membrane fusion (Naeve & Williams, 1990, EMBO J. 9:3857-3866). However, this finding has not been supported by other workers using either H2, H3 or H7 subtype HAs (Naim et al., 1992, J. Virol. 66:7585-7588; Simpson & Lamb, 1992, J. Virol. 66:790-803; Steinhauer et al., 1991, Virology 184:445-448; Veit et al., 1991, J. Virol. 65:2491-2500). It has been shown that substitution of the conserved cysteines at positions 553, 560 or 563 by serine (H2 and H3) or alanine (H7) did not significantly affect HA biosynthesis, intracellular transport or receptor-binding activity, when expressed from recombinant plasmid DNA or SV40 vectors (Doyle et al., 1985, J. Cell Biol. 100:704-714; Lazarovits & Roth, 1988, Cell 53:743-752; Naeve & Williams, 1990, EMBO J. 9:3857-3866; Naim et al., 1992, J. Virol. 66:7585-7588; Simpson & Lamb, 1992, J. Virol. 66:790-803; Steinhauer et al., 1991, Virology 184:445-448; Veit et al., 1991, J. Virol. 65:2491-2500). In fact, HA proteins with mutations in two (Simpson & Lamb, 1992, J. Virol. 66:790-803) or all three (Naim et al., 1992, J. Virol. 66:7585-7588) palmitate addition sites complemented an influenza virus with a temperature-sensitive mutation in the HA.
Recently, Naim and Roth (Naim & Roth, 1993, J. Virol. 67:4831-4841) have extended their studies of the role of the cytoplasmic tail by expressing HA variants from recombinant SV40 viruses in influenza virus-infected cells. Although chimeric HAs with foreign cytoplasmic sequences were efficiently excluded from viral envelopes, HAs which lacked a cytoplasmic tail were incorporated into virions. Similarly, Simpson and Lamb (Simpson & Lamb, 1992, J. Virol. 66:790-803) showed that HA mutants lacking a cytoplasmic tail were incorporated into viral particles, but the particles were found to be non-infectious. This would indicate that the conserved cytoplasmic sequences are not required for the incorporation of HA into virions but that the cytoplasmic tails of the HAs are necessary for the infectivity of the virus. However, data presented here using a different assay system indicate a role for these cysteine residues in the formation of infectious influenza virus.